Biochemical and molecular characterization of a novel metalloprotease from Pseudomonas fluorescens strain TBS09

• Published in: International journal of biological macromolecules Vol. 107; no. Pt B; pp. 2351 - 2363
• Main Authors: Boulkour Touioui, Souraya, Zaraî Jaouadi, Nadia, Bouacem, Khelifa, Ben Ayed, Rayda, Rekik, Hatem, Zenati, Bilal, Kourdali, Sidali, Boudjella, Hadjira, Sabaou, Nasserdine, Bejar, Samir, El Hattab, Mouhamed, Badis, Abdelmalek, Annane, Rachid, Jaouadi, Bassem
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01-02-2018
• Subjects: Amino Acid Sequence - genetics; Cloning, Molecular; Escherichia coli - genetics; Metalloproteases - chemistry; Metalloproteases - genetics; Metalloproteases - isolation & purification; Protease Inhibitors - chemistry; Protease Inhibitors - pharmacology; Pseudomonas fluorescens - enzymology; Pseudomonas fluorescens - genetics; Pseudomonas-metalloproteases; Recombinant enzyme; Sequence Analysis, DNA; Serralysin family; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Substrate Specificity; Serralysin family; Recombinant enzyme; Pseudomonas-metalloproteases
• ISSN: 0141-8130; 1879-0003
• DOI: 10.1016/j.ijbiomac.2017.10.116

•A novel extracellular P. fluorescens protease is purified (MPDZ) and characterized.•MPDZ is a metalloprotease and a monomer with a molecular mass of 50013.17Da.•Optimum pH and temperature values for activity were pH 7 and 60°C, respectively.•mpDZ gene encoding MPDZ is cloned, sequenced, and expressed in E. coli strain.•Enzymatic properties of the purified recombinant rMPDZ are similar to those of MPDZ.
A novel extracellular protease called MPDZ was purified and characterized from Pseudomonas fluorescens strain TBS09. The enzymatic properties of MPDZ were investigated using biochemical and biophysical methods. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI–TOF/MS) analysis revealed that it was a monomer with a molecular mass of 50013.17Da. The NH2-terminal 27 amino acid sequence of MPDZ showed high homology with those of Pseudomonas-proteases of the serralysin family. MPDZ showed optimal activity at pH 7 and 60°C. It was totally inhibited by EGTA, EDTA, and 1,10-phenanthroline, suggesting its belonging to the metalloprotease family. Because of the interesting properties, the mpDZ gene encoding MPDZ was cloned, sequenced, and expressed in E. coli. The deduced amino acid sequence showed a strong homology with other Pseudomonas-metalloproteases. The highest sequence identity value (97%) was obtained with AprX from P. fluorescens strain CY091, with only 12 different amino acid residues. The physico-chemical properties of the extracellular purified recombinant enzyme (rMPDZ) were similar to those of MPDZ. Overall, MPDZ is bestowed with a number of promising biochemical properties that might give new opportunities for its biocatalytic applications. These data constitute an essential first step towards an understanding of the properties of MPDZ enzyme.